Post-transplant thrombocytopenia is a major concern in clinical oncology which can delay additional therapy, require platelet transfusions at an increased treatment cost, and cause significant morbidity and occasional mortality. The most effective therapy for post-transplant thrombocytopenia is platelet transfusion. The use of high dose chemotherapy has greatly increased the demand for platelet products. Increasing the number of infused megakaryocytes (MK) and megakaryocytic progenitors (CFU- MK) through ex vivo expansion has the potential to decrease post- transplant thrombocytopenia. In the proposed studies, conditions for MK and CFU-MK expansion from unselected mPBSC will be optimized in a small-scale culture system. MK output will be assessed by cell morphology, flow cytometry (CD34, CD61, CD41, ploidy), and colony assay. The effects of several culture parameters including cytokine combination, serum-free medium, inoculation density, culture duration, and oxygenation on MK and CFU-MK yields will be examined. The identification of optimized MK expansion conditions will lead to the validation of this process at the clinical-scale in the AastromReplicell(TM) System, an automated GMP device. Based on Phase l results, Phase Il studies will include further optimization of clinical-scale MK production and the initiation of clinical augmentation trials to evaluate the effects of increased MK infusion on platelet engraftment in autologous transplant recipients. PROPOSED COMMERCIAL APPLICATIONS: An automated, GMP system capable of MK ex vivo expansion has significant clinical value. A MK-enriched cell product may alleviate post- transplant thrombocytopenia, significantly improving patient recovery. Reductions in platelet transfusions will reduce patient alloimmunization and treatment costs, and curtail the demand for platelet products, easing the increasing costs and safety concerns involved in donor recruitment and platelet collection. Clinical benefits of ex vivo generated MK in a transplant setting would lead to the commercialization of automated hardware, single use disposables, and reagent kits.